Neuronal degeneration is the cause of debilitating visual impairment associated with prevalent ocular diseases, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), retinoschisis, lattic degeneration, retinal detachment, and glaucoma. Other causes of visual impairment include artery or vein occlusion and diabetic retinopathy. Current treatment of these diseases remains unsatisfactory.
The identification and characterization of neural and retinal progenitor cells has opened new pathways for treating diseases associated with neuronal degeneration. Progenitor cells may help to restore vision in patients who have these diseases, by repopulating the damaged retina and/or by rescuing retinal neurons from further degeneration. In cellular replacement therapies, retinal progenitor cells or differentiated progeny are transplanted to replace diseased tissue. Cell replacement therapy may replace damaged cells with cultured stem/progenitor cells, or with endogenous stem/progenitor cells. Alternatively, genetically engineered stem or progenitor cells can be used to target gene products to sites of degeneration. These gene products can include survival-promoting factors to rescue degenerating neurons, factors that can act in an autocrine manner to promote survival and differentiation of grafted cells into site-specific neurons or to deliver neurotransmitter(s) to permit functional recovery.
Progenitor cells of the neural retina have been described as giving rise to all the neurons, photoreceptors, and the Muller glia of the eye. These progenitor cells have a simple bipolar morphology, and in most cases undergo their mitotic divisions at the ventricular surface. Immediately after their final mitotic division, one or both of the daughter cells begin to express characteristics of differentiating neurons. In the early embryonic retina, many of the divisions of the progenitor cells are symmetric; both progeny of a particular division can remain progenitor cells and continue to divide, although some of the mitotic divisions are asymmetric, with a particular division yielding a neuron and another progenitor cell, or two neurons of different types.
The lineages of the various types of retinal neurons share a common progenitor. Progeny of a single cell have been shown to include many different types of retinal neurons, and some clones contain a combination of both retinal neurons and Muller glia, the intrinsic glial cell of the retina. Progenitor cells can give rise to a neuron and a glial cell, two glia, two of the same type of neurons, or two different types of neurons. Thus, there is no strict lineage relationships among the different types of retinal cells.
Retinal progenitor cells have been isolated from embryonic tissues and grown in culture (Reh and Kljavin, 1989). Such cultures typically include growth factors such as epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). Peptides that stimulate receptor tyrosine kinases (e.g. FGF), serine threonine kinases (TGF-β3), and the hedgehog signaling pathway (Shh) can all influence the types of neurons that differentiate in the cultures. Thus, several different signaling systems are involved in the specification of cell identity and cell-specific gene expression. Many factors have opposing effects on the generation of the different retinal cell types; for example, TGF-α stimulates amacrine cell production but inhibits rod photoreceptor differentiation, while retinoic acid stimulates the progenitor to produce rods but prevents additional amacrine cells from differentiating in the culture. A large number of different types of factors, including extracellular matrix molecules such as S-laminin, and highly diffusible factors such as retinoic acid have been shown to affect photoreceptor differentiation, while fewer types of factors have been shown to have effects on the development of the other cell types.
The expansion of primary cultures of human retinal progenitors has potential for providing a source for transplantation to treat degenerative conditions of the retina such as macular degeneration and retinitis pigmentosa. However, it is difficult to obtain sufficient quantities of these cells from primary tissue. The ability to differentiate human retinal progenitors from stem cells would be of great interest for these purposes. The present invention addresses this issue.